The specific aim of this pilot study is to test the feasibility of using a polymer-based bioseparation process as a means of isolating the putative hyaluronate receptor CD44 from cell-membrane rich lysate, and subsequently to verify CD44's ability to bind hyaluronate. Hyaluronate receptor isolation is an initial step toward receptor characterization and fundamental to the long term objectives of our research; namely, to define hyaluronate metabolism at a cellular level.The centrality of hyaluronate to various biological processes, and the increasing recognition of its therapeutic applications provide justification for the proposed investigation. Poly-N-isopropylacrylamide (polyNIPAAm), a water soluble, thermally precipitating synthetic polymer, will be conjugated to the monoclonal anti-CD44 antibody (BU52) and utilized for bioseparation. The polymer-BU52 conjugate will be mixed and incubated separately with the cell-membrane rich fraction obtained from 2 cell lines known to contain CD44: peripheral blood lymphocytes and human dermal fibroblasts. Following complexation, the mixture will be gently heated above the critical temperature of 31 degrees C to precipitate the p(NIPAAm)-BU52-CD44 complex. The eluted fraction containing the presumptive CD44 will be electrophoresed and subjected to Western blot analysis to confirm the presence of CD44. Quantitative verification of the specific binding of hyaluronate to the extracted CD44 will be obtained by a binding analysis utilizing a dot blot assay and [3H]-hyaluronate. Non-specific binding will be assessed by a parallel incubation in the presence of a 100-fold excess of non- radioactive hyaluronate [3H]-hyaluronate binding to the receptor isolate will be determined by scintillation analysis. Differences in binding capacity will be analyzed using appropriate statistical analysis. The ability to isolate the hyaluronate receptor is an important prelude to it's characterization and will form the basis for a larger grant proposal (RO1) investigating hyaluronate metabolism at the cellular level. By exploiting the properties of the poly(NIPAAm) polymer, we seek to develop a continuous and rapid affinity precipitation bioseparation process capable of separating adequate quantities of receptor isolates. On a more general scale, we anticipate that the affinity precipitation separation system developed could be utilized in the analysis of other receptor systems,. The ability to evaluate receptor characteristics in isolation would permit a clearer distinction between receptor and nonreceptor interactions and has interesting therapeutic implications.